Toxoplasma gondii is an obligate intracellular protozoan which can invade and replicate within any nucleated cell. Intracellularly, T. gondii resides. within a parasitophorous vacuole, enclosed by the parasitophorous vacuole membrane (PVM). This specialized membrane prevents interaction of the vacuole with endocytic vesicular traffic of the host cell. Nonetheless, transport of nutrients and metabolites across the PVM is necessary for parasite survival, and a pore which allows the passage of small molecules is present in the PVM. In addition, host cell mitochondria and endoplasmic reticulum are tightly apposed to the PVM, presumably through interactions with PVM proteins. Determining the mechanisms by which such divergent functions are subserved by PVM components has important implications for understanding the cell biology of intracellular infection with T. gondii in particular and intracellular parasitism in general. T. gondii parasites contain a characteristic set of exocytic organelles, the rhoptries, dense granules and micronemes, which discharge at differential times during and after cell invasion. Selected proteins (the rhoptry components ROP2,3,4 and the dense granule protein GRA3) from these organelles are inserted into the PVM. Although the mechanisms and functional consequences of these targeting and insertion events are not yet defined, rhoptry and dense granule proteins associated with the PVM are certain to have important roles in the intracellular survival and development of the parasite. The long term objectives of our work are to determine the role that parasite components play in the overall process of invasion and intracellular replication by T. gondii. The specific aims of this project are to: 1.) Determine the transmembrane orientation of the T. gondii rhoptry proteins, ROP2,3, 4 within the PVM, using cDNA clones, antibody probes and a recently developed T. gondii transfection system, 2.) Define the mechanism for association of the dense granule protein GRA3 with the PVM, using both a cell free system and parasites transfected with GRA3, 3.) Characterize the properties and composition of the pore across the PVM, by microinjection of fluorescent tracers into infected cells and by reconstitution of the PVM pore into planar lipid bilayers, and 4.) Elucidate the mechanism of association of host cell mitochondria and endoplasmic reticulum with the PVM, using cell free systems and covalent cross-linking reagents to identify proteins mediating the interactions. T. gondii is the most common cause of focal central nervous system infections in patients with the Acquired Immunodeficiency Syndrome, and a devastating cause of congenital birth defects in infants born to mothers infected with T. gondii during pregnancy. The results of the proposed studies will help define the mechanism by which T. gondii causes disease and will facilitate the development of novel approaches and new targets for therapy of T. gondii infections.